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March 5, 2016

Oil-Dry Corporation of America Applies for Patent
to Stop AHPND/EMS

 

In USA patent application #20160030475, Oil-Dry Corporation of American, a developer, manufacture and marketer of absorbent products, describes a clay blend of yeast and glutamate to help decrease the effects of acute hepatopancreatic necrosis disease (AHPND), also known as early mortality syndrome (EMS), in shrimp.

 

The preferred embodiment of this product may be 100% “Calibrin®-Z”, a calcium montmorillonite clay, which has been heated to a temperature to decrease moisture and ground to a fine particle size.  This processing, heating to between 400°-800° C., and/or fine grinding (average particle size of 32-36 μm) has been shown to increase the toxin binding ability of the clay across multiple fungal and bacterial toxins.

 

Fifty percent of the shrimp feed produced in Mexico in 2015 contained Calibrin-Z®, a bacterial-toxin-control product that protects the hepatopancreas from the damaging effects of EMS.  Calibrin-Z® works by adsorbing the Vibrio parahaemolyticus bacterial toxin in the shrimp’s digestive system, thereby increasing the rate of survival in a shrimp crop.  Studies have shown improvements in survivability of up to 84% versus controls when shrimp challenged with the Vibrio parahaemolyticus toxin were fed Calibrin-Z®.  Information: Kevin Hayes, Calibrin-Z®, Amlan International, 410 North Michigan Avenue, Suite 400, Chicago, Illinois 60611, USA (phone 1-816-341-0713, email kevin.Hayes@oildri.com webpage http://www.amlan.com).

 

Here are some excerpts from the patent application:

 

“In another preferred embodiment, the clay, or clay, yeast and glutamate mixture of the invention may be formulated for addition to the water in which the shrimp are raised.”

 

 

Study One

(See the link in the source below for tabular data)

 

“A study was conducted to examine the effects of two products on the mortality caused by EMS in shrimp. The products were products that had previously been shown to reduce the effect of the bacterial enteric disease necrotic enteritis in chickens.  The products were: Product A) a 100% clay product, Calibrin®-Z (A); and Product B) was a blend of the clay, a yeast product, and monosodium glutamate (B).  These products were all tested at two concentrations, 0.25% and 0.5% of the diet.”

 

“Shrimp fed a diet supplemented with a clay or a combination of clay, a yeast product, and monosodium glutamate and infected with Vibrio parahaemolyticus culture to induce EMS/AHPND showed reduced mortality.  Survival at the end of the study in shrimp fed Product A at 0.5% was 95% and in those fed Product A at 0.25% was 85%.  Those fed Product B at 0.5% inclusion had a 52.5% survival rate and those shrimp fed Product B at a 0.25% inclusion rate had a 50% survival rate.  Survival in the group fed the control diet and not infected with V. parahaemolyticus was 92.5% while those fed the control diet and infected had a survival rate of 7.5%.  Histological examination showed that shrimp fed diets containing Product A showed no hepatopancreas lesions.”

 

“Juvenile Penaeus...vannamei were fed one of four diets containing proprietary products supplied by Oil-Dri Corporation of America for 7 days prior to oral exposure to Vibrio parahaemolyticus (agent causing Acute Hepatopancreatic Necrosis Disease) to determine if the products would have an effect on survival.”

 

“Survival at termination of the study in the group fed Product A at a 0.5% inclusion rate was 95% and survival in the group fed Product A at a 0.25% inclusion rate was 85%.  Termination survival in the group Product B at a 0.5% inclusion rate was 52.5% and survival in the group fed Product B at a 0.25% inclusion rate was 50%.  Survival in the positive control group, fed a control diet, was 7.5% and survival in the negative control group, also fed the control diet, was 92.5%.”

 

“The animals utilized in this study were originally obtained from Shrimp Improvement Systems.  A total of 240 SPF (specific pathogen free) P. vannamei were transferred from UAZ’s West Campus SPF facility and stocked into twelve 90 L aquaria at 20 animals per tank.”

 

“Eight tanks were fed diets formulated with the products (2 tanks for each diet).  Two tanks were designated as positive controls and two tanks served as negative environmental controls.  The positive control tanks were fed a commercially pelleted shrimp diet (Rangen, Inc., 40% protein) and were challenged with Vibrio parahaemolyticus to ensure that the challenge method worked and as a comparison for survival.  The negative control tanks were also fed the commercially pelleted control diet, but were not challenged with Vibrio parahaemolyticus.”

 

“All tanks were fed their respective diets at 5% bodyweight once a day for the duration of the study.  All aquaria were outfitted with an oyster-shell filter, aeration and were covered with a plastic sheet to reduce the risk of cross contamination.  The negative control tanks were kept isolated in a separate building and fed before the Vibrio parahaemolyticus challenge tanks.”

 

“Two products were used in this study.  The first product was labeled as Product A and the second was labeled as Product B.  The four diets included:

 

Product A—0.5% inclusion rate

Product A—0.25% inclusion rate

Product B—0.5% inclusion rate

Product B—0.25% inclusion rate”

 

“The test materials were combined with Rangen (Rangen Inc., 115 13 Ave. So. Buhl, Id.) 40% protein commercial shrimp premix, 40% water, and the binder carboxymethyl cellulose at 3% inclusion rate, and then cold extruded through a meat grinder.  The resulting feed was dried overnight at 40° C. and then broken into an appropriately sized pellet.”

 

“Each tank was fed the appropriate diet for a total of 7 days prior to AHPND challenge.  Once the AHPND study began, all tanks were checked daily for moribund or dead animals.  A few moribund animals were preserved in Davidson’s AFA fixative and processed for routine histology.  All mortalities not preserved by fixation and all dead animals were removed from the tanks and frozen.  At termination of the study, two live animals from each tank were preserved in Davidson’s AFA fixative and all remaining animals were frozen.  The study was terminated 15 days post initial exposure to AHPND.”

 

On day 0 of the AHPND challenge portion of the study, all challenge aquaria were fed the appropriate diet (either Products A or B or the control diet) that had been soaked in a broth containing the AHPND inducing V. parahaemolyticus at an optical density of 1.57.  No mortality was noted in any tank by day 1 post infection (p.i.), so all tanks were re-challenged on day 2 p.i. in the same manner as on day 0, but this time with a broth at an optical density of 1.26.”

 

“By day 7 p.i., no mortality had been noted in any tank with the exception of one of the positive control tanks, so the tanks were again re-challenged, but this time they were given two feedings in a single day.  On day 8 p.i. of the AHPND challenge, all AHPND challenge aquaria were fed a commercially pelleted shrimp diet (Rangen Inc., 35% protein) which had been soaked in an AHPND-causing Vibrio parahaemolyticus broth with an optical density of 1.88.  On the afternoon of day 8 p.i., all AHPND challenge aquaria were fed a second dose of the same Vibrio parahaemolyticus broth, but this time the broth was added to the respective Oil-Dri diet prior to feeding.”

 

“No dead or moribund animals were noted in the negative control tanks during the study, although a reduction in numbers was noted and the missing animals were attributed to cannibalism.  At termination of the study 15 days post-infection (22 days total), a total of 20 live animals out of 20 stocked on day 0 were collected from the first tank and 17 live animals out of 20 were collected from the second tank.  Combined termination survival was 92.5%, with individual survival rates of 100% and 85%, respectively.”

 

“Diet 1 (Product A at a 0.5% inclusion rate): On day 9 post EMS exposure, one moribund animal was noted in one of the challenge tanks fed Diet 1.  No other dead or moribund animals were noted in this group of tanks, although a reduction in numbers was noted in one tank by day 13 post-infection.  At termination of the study on day 15 post-infection, a total of 18 live animals were collected from the first tank and 20 live animals were collected from the second tank, resulting in survival rates of 90% and 100%, respectively.  Combined survival for this group was 95%.”

 

“Diet 2 (Product A at a 0.25% inclusion rate): No dead or moribund animals were noted in either tank fed Diet 2 during the study, although a reduction in numbers was noted on day 13 post-infection.  The missing animals were likely weak and fully cannibalized before they could be removed from the tank.  At termination of the study, 18 live animals were collected from the first tank and 16 animals were collected from the second tank resulting in tank survival rates of 90% and 80%, respectively.  Combined survival for this group was 85%.”

 

“Diet 3 (Product B at a 0.5% inclusion rate): The first dead and moribund animals were noted in one of the tanks fed Diet 3 on day 10 post-exposure (2 days after final exposure).  Dead and moribund animals were collected from the first tank daily until day 12 p.i., when mortality ceased.  No dead or moribund animals were collected from the second tank during the study, although a reduction in numbers was noted on day 13 post-infection.  At termination of the study 3 live animals were collected from the first tank, resulting in a 15% survival rate and 18 live animals were collected from the second tank, resulting in a 90% survival rate.  Combined termination survival for this group was 52.5%.”

 

“Diet 4 (Product B at a 0.25% inclusion rate): The first dead and moribund animals were noted in both tanks fed Diet 4 on day 9 post EMS exposure.  Dead animals were collected daily until mortality ceased on day 11 post-exposure.  At termination of the study 4 live animals were collected from one tank, resulting in a 20% survival rate and 16 animals were collected from the second tank, resulting in an 80% survival rate.  Combined termination survival for this group was 50%.”

 

“AHPND Positive Control Group (Control diet): The first dead animals were noted in one of the P. vannamei AHPND positive control tanks on day 3 post-infection.  Dead and moribund animals were collected daily from the first tank until mortality ceased on day 9 post-infection.  The first dead animals were noted in the second tank on day 9 post-infection and continuing until day 10 when mortality ceased.  At termination of the study, a total of 2 live animals were collected from the first tank, resulting in a 5% survival rate and 2 live animals were collected from the second tank, resulting in a 10% survival rate.  Combined survival in the positive control group was 7.5%.”

 

 

Study Two with Calibrin-Z®

(See the link in the source below for tabular data)

 

“The study was repeated using Calibrin-Z® since it had previously shown to have the most beneficial effect on mortality caused by early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp in the previous study.  This product was tested at two concentrations, 0.25% and 0.5% of the diet.”

 

“Shrimp fed a diet supplemented with Calibrin-Z® and infected with Vibrio parahaemolyticus culture to induce EMS/AHPND showed reduced mortality compared to a challenged group that was not fed Calibrin-Z®.  Survival at the termination of the study in the group fed Calibrin®-Z at a 0.25% inclusion rate was 66.7% and survival in the group fed Calibrin-Z® at a 0.5% inclusion rate was 39.8%.  Survival in the positive control group, the group challenged with V. parahaemolyticus and fed the control diet, was 1.5% and survival in the negative (unchallenged) control group, also fed the control diet, was 100%.  Histological examination showed that shrimp fed diets containing Calibrin®-Z showed no hepatopancreas lesions.”

 

“Juvenile Penaeus...vannamei were fed one of two diets (0.25 or 0.5% inclusion) containing a proprietary product supplied by Oil-Dri Corporation of America for 7 days prior to oral exposure to Vibrio parahaemolyticus (agent causing AHPND) to determine if the product would have an effect on survival.

 

“Survival at the end of the study in the group fed Calibrin®-Z at a 0.25% inclusion rate was 66.7% and those fed Calibrin®-Z at a 0.5% inclusion rate had a 39.8% survival rate.  Survival in the positive control group, fed a control diet, was 1.5% and survival in the negative control group (the only unchallenged group), also fed the control diet, was 100%.”

 

“The animals utilized in this study were originally obtained from Shrimp Improvement Systems.  A total of 264 SPF (specific pathogen free) P. vannamei were transferred from University of Arizona's West Campus SPF facility and stocked into twelve 90 L aquaria at 22 animals per tank.”

 

“Six tanks were fed diets formulated with the product (3 tanks for each diet).  Three tanks were designated as positive controls and three tanks served as negative environmental controls.  The positive control tanks were fed a commercially pelleted shrimp diet and were challenged with Vibrio parahaemolyticus to ensure that the challenge method worked and as a comparison for survival.  The negative control tanks were also fed the commercially pelleted control diet, but were not challenged with Vibrio parahaemolyticus.  One negative control tank was set up next to the AHPND challenge tanks and the two remaining tanks were kept isolated in a separate building.”

 

“All tanks were fed their respective diets at 5% bodyweight once a day for the duration of the study.  All aquaria were outfitted with an oyster-shell filter, aeration and were covered with a plastic sheet to reduce the risk of cross contamination. All negative control tanks were kept isolated in a separate building and fed before the Vibrio parahaemolyticus challenge tanks.”

 

“Product Calibrin-Z® was used in this study.  The three diets made included: Diet 1—Calibrin®-Z at 0.25% inclusion rate, Diet 2—Calibrin-Z® at 0.50% inclusion rate and Diet 3—Control Diet (no Calibrin-Z®).”

 

“The test materials were combined with Rangen (Rangen Inc., 115 13 Ave. So. Buhl, Id.) 40% protein commercial shrimp premix, 40% water, and the binder carboxymethyl cellulose at 3% inclusion rate, and then cold extruded through a meat grinder.  The resulting feed was dried overnight at 40° C. and then broken into an appropriately sized pellet.”

 

“Each tank was fed the appropriate diet for a total of 7 days prior to AHPND challenge.  Once the AHPND study began, all tanks were checked daily for moribund or dead animals.  A few moribund animals were preserved in Davidson's AFA fixative and processed for routine histology.  All mortalities not preserved by fixation and all dead animals were removed from the tanks and frozen.  At termination of the study, 1-2 live survivors from each tank were preserved in Davidson's AFA fixative and any remaining animals were frozen.  The study was terminated 7 days post initial exposure to AHPND.”

 

“Different from the previous study, an 18-20 hour culture of Vibrio parahaemolyticus which causes AHPND (Vietnam isolate) with a reduced optical density of 1.5 was utilized in this study.  On day 0 of the AHPND challenge portion of the study, all challenge aquaria were fed a commercially pelleted shrimp diet (Rangen Inc., 40% protein) which had been soaked in a broth containing the AHPND inducing V.  parahaemolyticus at an optical density of 0.071 with 5.0×105 colony-forming units.  On the afternoon of day 0 post infection all AHPND challenge aquaria were fed a second dose of the same Vibrio parahaemolyticus broth, but this time the broth was added to the respective diets prior to feeding.  That same day, all animals in each tank were counted to obtain an actual day 0 start number for the AHPND challenge.”

 

“No mortality was noted in any of the challenge tanks by day 2 post infection, so all tanks were re-challenged on day 3 post-infection in the same manner as on day 0, but this time with a broth at an optical density of 1.75 with 1.1×109 colony forming units.”

 

“No dead or moribund animals were noted in the negative control tanks during the study.  At termination of the study 10 days post-infection, a total of 21 live animals out of 21 counted on day 0 post-infection were collected from all three negative control tanks, resulting in individual and combined survival rates of 100%.”

 

“Diet 1 (Calibrin® Z at a 0.25% inclusion rate): The first dead animals were noted in one of the challenge tanks fed Diet 1 beginning on day 4 post initial challenge.  Dead and moribund animals were collected from that tank daily until the last animal died on day 6 post infection.  No dead or moribund animals were noted in the remaining two tanks during the challenge study.  At termination of the study on day 10 post-infection, no survivors were collected from the first tank and 19 and 20 survivors were collected from the remaining two tanks, resulting in survival rates of 0% and 100%, respectively.  Combined survival for this group was 66.7%.”

 

“Diet 2 (Calibrin® Z at a 0.5% inclusion rate): The first dead and moribund animals were noted in the two tanks fed Diet 2 beginning on day 3 post-infection.  Dead and moribund animals were collected daily from those two tanks until mortality ceased on day 7 post-infection.”

 

“No dead or moribund animals were noted in the final tank in this group during the challenge study.  At termination of the study, 3 live animals were collected from the first tank, 1 animal was collected from the second tank and 20 live animals were collected from the final tank.  Tank survival rates were 14%, 5% and 100%, respectively.  Combined survival for this group was 39.8%.”

 

“AHPND Challenge Group (Control diet): The first dead animals were noted in one of the AHPND positive control tanks on day 3 post-infection.  The first dead animals were noted in the second tank on day 4 post-infection and in the third tank on day 5 post-infection.  Dead and moribund animals were collected daily from all three tanks until mortality ceased on day 8 post-infection.  At termination of the study, no live animals were collected from two tanks and 1 live animal was collected from the last tank, resulting in a 0% survival rates for two tanks and a 4% survival rate for the last tank.  Combined survival in the positive control group was 1.5%.”

 

Information: San Ching, Fang Chi and Ron Cravens.  Oil-Dry Corporation of American, 410 North Michigan Avenue, Suite 400, Chicago, Illinois 60611, USA (Phone 1-312-321-1514, Fax 1-312-321-9525, Email customer.service@uline.com, Webpage http://www.oildri.net).

 

Sources: 1.  FPO (Free Patents Online).  Use of a Clay Product or a Clay Blend Product to Decrease the Effects of Bacterial Disease in Shrimp.  San Ching, Fang Chi and Ron Cravens.  February 4, 2016. 2. Bob Rosenberry, Shrimp News International, March 5, 2016.

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